Effects of leucine supplementation and resistance exercise on dexamethasone-induced muscle atrophy and insulin resistance in rats

Objective: We aimed to evaluate the effects of resistance exercise (RE) and leucine (LEU) supplementation on dexamethasone (DEXA)-induced muscle atrophy and insulin resistance. Methods: Male Wistar rats were randomly divided into DEXA(DEX), DEXA + RE (DEX-RE), DEXA + LEU (DEX-LEU), and DEXA + RE + LEU (DEX-RE-LEU) groups. Each group received DEXA 5 mg . kg(-1) . d(-1) for 7 d from drinking water and were pair-fed to the DEX group; LEU-supplemented groups received 0.135 g . kg(-1) . d(-1) through gavage for 7 d; the RE protocol was based on three sessions of squat-type exercise composed by three sets of 10 repetitions at 70% of maximal voluntary strength capacity. Results: The plantaris mass was significantly greater in both trained groups compared with the non-trained groups. Muscle cross-sectional area and fiber areas did not differ between groups. Both trained groups displayed significant increases in the number of intermediated fibers (IIa/IIx), a decreased number of fast-twitch fibers (IIb), an increased ratio of the proteins phospho(Ser2448)/ total mammalian target of rapamycin and phospho(Thr389)/total 70-kDa ribosomal protein S6 kinase. and a decreased ratio of phospho(Ser253)/total Forkhead box protein-3a. Plasma glucose was significantly increased in the DEX-LEU group compared with the DEX group and RE significantly decreased hyperglycemia. The DEX-LEU group displayed decreased glucose transporter-4 translocation compared with the DEX group and RE restored this response. LEU supplementation worsened insulin sensitivity and did not attenuate muscle wasting in rats treated with DEXA. Conversely, RE modulated glucose homeostasis and fiber type transition in the plantaris muscle. Conclusion: Resistance exercise but not LEU supplementation promoted fiber type transition and improved glucose homeostasis in DEXA-treated rats. (C) 2012 Elsevier Inc. All rights reserved.

Long-term leucine supplementation reduces fat mass gain without changing body protein status of aging rats

Objective: Aging is characterized by alterations in body composition such as an increase in body fat and decreases in muscle mass (sarcopenia) and bone density (osteopenia). Leucine supplementation has been shown to acutely stimulate protein synthesis and to decrease body fat. However, the long-term effect of consistent leucine supplementation is not well defined. This study investigated the effect of leucine supplementation during aging. Methods: Six-month-old rats were divided into three groups: an adult group (n = 10) euthanized at 6 mo of age, a leucine group (n = 16) that received a diet supplemented with 4% leucine for 40 wk, and a control group (n = 19) that received the control diet for 40 wk. The following parameters were evaluated: body weight, food intake, chemical carcass composition, indicators of acquired chronic diseases, and indicators of protein nutritional status. Results: Body weight and fat were lower in the leucine group after 40 wk of supplementation compared with the control group but still higher than in the adult group. The lipid and glycemic profiles were equally altered in the control and leucine groups because of aging. In addition, leucine supplementation did not affect the changes in protein status parameters associated with aging, such as decreases in body and muscle protein and total serum protein. Conclusion: The results indicate that leucine supplementation attenuates body fat gain during aging but does not affect risk indicators of acquired chronic diseases. Furthermore, supplemented animals did not show signs of a prevention of the decrease in lean mass associated with aging. (C) 2012 Elsevier Inc. All rights reserved.

Leucine supplementation combined with resistance exercise improves the plasma lipid profile of dexamethasone-treated rats

The impact of leucine supplementation and resistance exercise (RE) on plasma lipid profile was evaluated in adult rats treated with dexamethasone, an experimental model of dyslipidemia. Total cholesterol did not differ among groups. Furthermore, leucine supplementation did not promote improvement in the plasma total cholesterol and LDL-c of the animals. However, plasma TG and VLDL-c were significantly decreased and HDL-c increased after 7 days of leucine supplementation combined with RE. In conclusion, leucine supplementation combined with RE, but not isolated, improved the plasma lipid profile of dexamethasone-induced dyslipidemic rats.

Dose and latency effects of leucine supplementation in modulating glucose homeostasis: opposite effects in healthy and glucocorticoid-induced insulin-resistance states

Dexamethasone (DEXA) is a potent immunosupressant and anti-inflammatory agent whose main side effects are muscle atrophy and insulin resistance in skeletal muscles. In this context, leucine supplementation may represent a way to limit the DEXA side effects. In this study, we have investigated the effects of a low and a high dose of leucine supplementation (via a bolus) on glucose homeostasis, muscle mass and muscle strength in energy-restricted and DEXA-treated rats. Since the leucine response may also be linked to the administration of this amino acid, we performed a second set of experiments with leucine given in bolus (via gavage) versus leucine given via drinking water. Leucine supplementation was found to produce positive effects (e. g., reduced insulin levels) only when administrated in low dosage, both via the bolus or via drinking water. However, under DEXA treatment, leucine administration was found to significantly influence this response, since leucine supplementation via drinking water clearly induced a diabetic state, whereas the same effect was not observed when supplied via the gavage.

Oral leucine supplementation is sensed by the brain but neither reduces food intake nor induces an anorectic pattern of gene expression in the hypothalamus

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity

The possible role of leucine in modulating glucose homeostasis under distinct catabolic conditions

Branched-chain amino acids (BCAA) (especially leucine) have been shown to activate protein synthesis pathways, decrease proteolysis and increase insulin sensitivity. Furthermore, it appears that leucine can be used as a nutritional therapy to avoid sarcopenia and skeletal muscle atrophy due to immobilization or glucocorticoid treatment. However, it is of note that all of these conditions are related to insulin resistance to varying degrees and affect different tissues, particularly skeletal muscle. Additionally, evidence from recent studies demonstrate that a combination of protein containing high levels of leucine with nutrients containing saturated fatty acids or an excess of leucine are capable of inducing insulin resistance. From this discussion, a few major questions arise. First, what is the role of a combination of macronutrients in inducing insulin resistance? Second, in insulin resistance, does leucine supplementation follow the same path observed under healthy conditions? Finally, what are the dose-dependent outcome and the latency of leucine effect under such conditions? The present article discusses these questions based on data from the literature and experiments performed by our group. (C) 2012 Elsevier Ltd. All rights reserved.

Potential antiproteolytic effects of L-leucine: observations of in vitro and in vivo studies

The purpose of present review is to describe the effect of leucine supplementation on skeletal muscle proteolysis suppression in both in vivo and in vitro studies. Most studies, using in vitro methodology, incubated skeletal muscles with leucine with different doses and the results suggests that there is a dose-dependent effect. The same responses can be observed in in vivo studies. Importantly, the leucine effects on skeletal muscle protein synthesis are not always connected to the inhibition of skeletal muscle proteolysis. As a matter of fact, high doses of leucine incubation can promote suppression of muscle proteolysis without additional effects on protein synthesis, and low leucine doses improve skeletal muscle protein ynthesis but have no effect on skeletal muscle proteolysis. These research findings may have an important clinical relevancy, because muscle loss in atrophic states would be reversed by specific leucine supplementation doses. Additionally, it has been clearly demonstrated that leucine administration suppresses skeletal muscle proteolysis in various catabolic states. Thus, if protein metabolism changes during different atrophic conditions, it is not surprising that the leucine dose-effect relationship must also change, according to atrophy or pathological state and catabolism magnitude. In conclusion, leucine has a potential role on attenuate skeletal muscle proteolysis. Future studies will help to sharpen the leucine efficacy on skeletal muscle protein degradation during several atrophic states.

Leucine and HMB differentially modulate proteasome system in skeletal muscle under different sarcopenic conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.

Metabolic and functional effects of beta-hydroxy-beta-methylbutyrate (HMB) supplementation in skeletal muscle

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite derived from leucine. The anti-catabolic effect of HMB is well documented but its effect upon skeletal muscle strength and fatigue is still uncertain. In the present study, male Wistar rats were supplemented with HMB (320 mg/kg per day) for 4 weeks. Placebo group received saline solution only. Muscle strength (twitch and tetanic force) and resistance to acute muscle fatigue of the gastrocnemius muscle were evaluated by direct electrical stimulation of the sciatic nerve. The content of ATP and glycogen in red and white portions of gastrocnemius muscle were also evaluated. The effect of HMB on citrate synthase (CS) activity was also investigated. Muscle tetanic force was increased by HMB supplementation. No change was observed in time to peak of contraction and relaxation time. Resistance to acute muscle fatigue during intense contractile activity was also improved after HMB supplementation. Glycogen content was increased in both white (by fivefold) and red (by fourfold) portions of gastrocnemius muscle. HMB supplementation also increased the ATP content in red (by twofold) and white (1.2-fold) portions of gastrocnemius muscle. CS activity was increased by twofold in red portion of gastrocnemius muscle. These results support the proposition that HMB supplementation have marked change in oxidative metabolism improving muscle strength generation and performance during intense contractions.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

Leucine Is Essential for Attenuating Fetal Growth Restriction Caused by a Protein-Restricted Diet in Rats

Certain amino acids, such as leucine (Leu) are not only substrates for protein synthesis but also are important regulators of protein metabolism. Moreover, it is known that alterations in intrauterine growth favor the development of chronic diseases in adulthood. Therefore, we investigated the role of Leu in combination with other BCAA on effects that are induced by maternal protein restriction on fetal growth. Wistar rats were divided into 4 groups according to the diet provided during pregnancy: control (C; 20% casein); V+I [5% casein + 2% L-valine (Val) + 2% L-isoleucine (Ile)1; KYT 15% casein + 1.8% L-lysine (Lys) + 1.2% L-tyrosine (Tyr) + 1% L-threonine (Thr)1; and BCAA (5% casein + 1.8% L-Leu + 1.2% L-Val + 1% L-Ile). Maternal protein restriction reduced the growth and organ weight of the offspring of dams receiving the V+I and KYT diets compared with the C group. Supplementation with BCAA reversed this growth deficit, minimizing the difference or restoring the mass of organs and carcass fat, the liver and muscle protein, and the RNA concentrations compared with newborns in the C group (P < 0.05). These effects could be explained by the activation of the mTOR signaling pathway, because phosphorylation of 4E-BP1 in the liver of offspring of the BCAA group was greater than that in the C, V+I, and KYT groups. The present results identify a critical role for Leu in association with other BCAA in the activation of the mTOR signaling pathway for the control of altered intrauterine growth induced by a maternal low-protein diet. J. Nutr. 142: 924-930, 2012.

Sunflower Oil Supplementation Has Proinflammatory Effects and Does Not Reverse Insulin Resistance in Obesity Induced by High-Fat Diet in C57BL/6Mice

High consumption of polyunsaturated fatty acids, such as sunflower oil has been associated to beneficial effects in plasma lipid profile, but its role on inflammation and insulin resistance is not fully elucidated yet. We evaluated the effect of sunflower oil supplementation on inflammatory state and insulin resistance condition in HFD-induced obese mice. C57BL/ 6 male mice (8 weeks) were divided in four groups: (a) control diet (CD), (b) HFD, (c) CD supplemented with n-6 (CD + n-6), and (d) HFD supplemented with n-6 (HFD + n-6). CD + n-6 and HFD + n-6 were supplemented with sunflower oil by oral gavage at 2 g/ Kg of body weight, three times per week. CD and HFD were supplemented with water instead at the same dose. HFD induced whole andmuscle-specific insulin resistance associated with increased inflammatory markers in insulin-sensitive tissues andmacrophage cells. Sunflower oil supplementation was not efficient in preventing or reducing these parameters. In addition, the supplementation increased pro-inflammatory cytokine production by macrophages and tissues. Lipid profile, on the other hand, was improved with the sunflower oil supplementation in animals fed HFD. In conclusion, sunflower oil supplementation improves lipid profile, but it does not prevent or attenuate insulin resistance and inflammation induced by HFD in C57BL/ 6 mice.

Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle

The effect of short-term creatine (Cr) supplementation upon content of skeletal muscle-derived-reactive oxygen species (ROS) was investigated. Wistar rats were supplemented with Cr (5 g/kg BW) or vehicle, by gavage, for 6 days. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated for evaluation of ROS content using Amplex-UltraRed reagent. The analysis of expression and activity of antioxidant enzymes (superoxide dismutase 1 and 2, catalase and glutathione peroxidase) were performed. Direct scavenger action of Cr on superoxide radical and hydrogen peroxide was also investigated. Short-term Cr supplementation attenuated ROS content in both soleus and EDL muscles (by 41 and 33.7%, respectively). Cr supplementation did not change expression and activity of antioxidant enzymes. Basal TBARS content was not altered by Cr supplementation. In cell-free experiments, Cr showed a scavenger effect on superoxide radical in concentrations of 20 and 40 mM, but not on hydrogen peroxide. These results indicate that Cr supplementation decreases ROS content in skeletal muscle possibly due to a direct action of Cr molecule on superoxide radical.

EFFECTS OF VITAMIN C SUPPLEMENTATION ON ACUTE PHASE CHAGAS DISEASE IN EXPERIMENTALLY INFECTED MICE WITH Trypanosoma cruzi QM1 STRAIN

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

Positive effects of zinc supplementation on growth, GH, IGF1, and IGFBP3 in eutrophic children

Zinc is an essential micronutrient for growth and development. Its deficiency causes growth retardation in children and adolescents. The present study analyzes the effect of zinc on growth hormone (GH) secretion, insulin-like growth factor 1 (IGF1), and insulin-like growth factor-binding protein 3 (IGFBP3) in normal children before puberty. Thirty normal children were studied, 15 boys and 15 girls, aged 6-9 years. They were orally supplemented with 5 mg Zn/day for 3 months and 0.06537 mg Zn/kg body weight was injected before and after oral supplementation. Dietary intake and anthropometric measurements were assessed at baseline and end of study. Plasma GH levels increased during intravenous zinc administration and IGF1 and IGFBP3 increased after oral zinc supplementation. There was a positive correlation between the areas under the curves of GH and zinc after oral supplementation. Zinc supplementation was possibly effective in improving the body zinc status of the children, secretory levels of IGF1 and IGFBP3, GH potentialization, and height.